Metabolic function of Corynebacterium glutamicum aminotransferases AlaT and AvtA and impact on L-valine production.
Aminotransferases (ATs) interacting with L-alanine are the least studied bacterial ATs. Whereas AlaT converts pyruvate to L-alanine in a glutamate-dependent reaction, AvtA is able to convert pyruvate to L-alanine in an L-valine-dependent manner. We show here that the wild type of Corynebacterium glutamicum with a deletion of either of the corresponding genes does not exhibit an explicit growth deficiency. However, a double mutant was auxotrophic for L-alanine, showing that both ATs can provide L-alanine and that they are the only ATs involved. Kinetic studies with isolated enzymes demonstrate that the catalytic efficiency, k(cat)/K(m), of AlaT is higher than 1 order of magnitude in the direction of L-alanine formation (3.5 x 10(4) M(-1) s(-1)), but no preference was apparent for AvtA, suggesting that AlaT is the principal L-alanine-supplying enzyme. This is in line with the cytosolic L-alanine concentration, which is reduced in the exponential growth phase from 95 mM to 18 mM by a deletion of alaT, whereas avtA deletion decreases the L-alanine concentration only to 76 mM. The combined data show that the presence of both ATs has subtle but obvious consequences on balancing intracellular amino acid pools in the wild type. The consequences are more obvious in an L-valine production strain where a high intracellular drain-off of the L-alanine precursor pyruvate prevails. We therefore used deletion of alaT to successfully reduce the contaminating L-alanine in extracellular accumulated L-valine by 80%.
Marienhagen
Jan
J
Institute of Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany.
Eggeling
Lothar
L
D000409:Alanine / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003600:Cytosol / Q000737:chemistry; D017353:Gene Deletion; D007700:Kinetics; D019289:Pyruvic Acid / Q000378:metabolism; D013379:Substrate Specificity; D000637:Transaminases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014633:Valine / Q000096:biosynthesis*
D019289:Pyruvic Acid; D000637:Transaminases; D014633:Valine; D000409:Alanine